Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Comparison of merits of DNA sequencing, PCR-SSCP and MFP assays in the detection of drug resistance in Mycobacterium leprae

Fan Long¹, JiuLong Li¹, Jiedeng Jia²

¹Department of Clinical Laboratory; ²Department of Orthopedics, The People's Hospital of Gaoping District, Nanchong, Sichuan637000, China.

For correspondence:- Jiedeng Jia Email: jiedengjia@hotmail.com Tel:+868173362777

Accepted: 20 July 2021 Published: 31 August 2021

Citation: Long F, Li J, Jia J. Comparison of merits of DNA sequencing, PCR-SSCP and MFP assays in the detection of drug resistance in Mycobacterium leprae. Trop J Pharm Res 2021; 20(8):1601-1607 doi: 10.4314/tjpr.v20i8.8

© 2021 The authors.
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Abstract

Purpose: To compare the merits of conventional Mouse Foot Pad (MFP) assay, Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) analysis, and Direct-DNA sequencing in identification of drug resistance in M. leprae.

Methods: A total of 41 leprosy cases were investigated for drug resistance using MFP assay, whereby growth results were obtained in 23 cases. The DNAs extracted from these samples were amplified in polymerase chain reaction (PCR) using specific primers in order to determine the drug resistance-loci. Furthermore, PCRSSCP analysis was carried out using PCR amplicons, while gel electrophoresis was performed to identify any shift in mobility in any one of the DNA strands, as well as the pattern of mutation.

Results: A total of 5 isolates exhibited the highest degree of resistance R100, whereas 4 isolates showed intermediate level of resistance R10. In contrast, the least resistance was seen in R1. There were no mutations in 4 out of 10 strains which were dapsone-resistant, i.e., 1 HR, 2 IR and 1 LR. Moreover, there was no mutation in 305 bp sequence of the rpoB gene. DNA sequencing sensitivity was 60 %, whereas the sensitive isolates tested in the experiments exhibited no mutation, resulting in 100 % specificity.

Conclusion: These results indicate the advantages of molecular techniques over conventional MFP technique. The study has revealed that PCR-SSCP analysis, a rapid, specific and less expensive method, has greater potential for use in routine testing of the susceptibility of M. leprae to rifampicin and dapsone, than the other tests.

Keywords: Molecular techniques, Mycobacterium leprae, Lapromatous, Leprosy, Drug resistance, Mouse Foot Pad (MFP) assay, Polymerase Chain Reaction-Single Stran